Predictive modeling for cow's milk allergy remission by low-dose oral immunotherapy in young children

Background The effectiveness of slow low-dose oral immunotherapy (SLOIT) for cow's milk (CM) allergy has been reported. Most OIT studies have discussed the target populations over 4 years old. Furthermore, no predicting modeling is reported for CM allergy remission by CM-SLOIT under 4 years of age. Objective We sought to develop a predictive model for CM allergy remission by SLOIT after 3 years in young children who started CM-SLOIT under 4 years of age. Methods We included young children with cow's milk allergy or cow's milk sensitization (development modeling set with 120 children and validation modeling set with 71 children). We did logistic regression analysis to develop the models. We calculated the area under the receiver operating curves (ROC-AUCs) to evaluate the predictive modeling performance. Results The model (CM-sIgE before SLOIT + age at beginning SLOIT + serum TARC before starting SLOIT + CM-sIgE titer one year after OIT) showed good discrimination with the ROC-AUC of 0.83 (95% CI:0.76–0.91) on internal validation. Applying the model to the validation set gave good discrimination (ROC-AUC = 0.89, 95% CI:0.80–0.97) and a reasonable calibration (intraclass correlation coefficient = 0.88, 95% CI:0.62–0.97). Conclusion We developed and validated predictive modeling for determining the remission rate of CM allergy at 3 years after SLOIT under 4 years of age in children with CM allergy. This predictive model is highly accurate and can support CM allergy management. (226 words)


INTRODUCTION
The food allergy epidemic is a global issue that imposes a heavy burden. 1 Cow's milk allergy is one of the most frequent causal food allergens in food allergy children. 2,3Absolute causal food avoidance delays food allergy tolerance induction. 4Furthermore, children of high IgE-sensitized cow's milk could not obtain natural remission with a lifelong burden. 5The Japanese food allergy guidelines have not recommended absolute causal food elimination but minimum causal food elimination based on the results of an oral food challenge to confirm the threshold. 6systematic review addressed that oral immunotherapy for cow's milk had highly contributed to food allergy treatment, but adverse allergic reactions were common. 7he guideline recommends oral immunotherapy for food allergy starting around 4-5 years old. 8Slow lowdose oral immunotherapy is one of the oral immunotherapy methods avoiding serious adverse reactions that proceed below the threshold level by inducing immune tolerance by starting with a small amount of allergen, slowly increasing the intake, and confirming an increase in the threshold level and continuing with partial elimination. 9,10e consider that slow, low-dose oral immunotherapy is also useful in infants.Taking even small amounts of allergenic foods could increase the thresholds and reduce allergic symptoms by accidental causal food intake.
A recent review indicated that oral immunotherapy was initiated in infants at the age of 3 months.There is no global harmonized definition of cow's milk tolerance.However, in Japan, the standardized definition for cow's milk complete remission is availability of 200 ml of cow's milk (6.6 g of cow's milk protein), and we use this definition in school lunch management. 6Children who can take 10 ml of cow's milk are usually able to take confectionery containing cow's milk.
There might be multiple factors influencing tolerance induction.Excellent eczema management itself could reduce IgE titers. 11Early introduction of allergenic foods could induce tolerance induction.However, affecting factors need to be betterrecognized.Caregivers want to know the future prognosis of slow low-dose oral immunotherapy in cow's milk.However, we have not had a predicting model for cow's milk tolerance by slow low-dose oral immunotherapy under four years of age.We sought to develop a predictive model for cow's milk tolerance by slow low-dose oral immunotherapy in the first three years in young children with cow's milk allergy who started under four.

Participants and study design
We collected medical chart records of the children with cow's milk sensitization or cow's milk allergy.Eligible populations were as follows: 1) the first visit from January 2014 and October 2018, 2) sensitized to cow's milk, 3) complete elimination for cow's milk and dairy products, 4) started slow low-dose oral immunotherapy 9 with cow's milk under 4 years of age, and 5) slow low-dose oral immunotherapy followed for 3 years.The slow, low-dose oral immunotherapy protocol details Fig. 1 A regimen of slow low-dose oral immunotherapy were shown in Fig. 1.Physicians decide the starting and maintenance doses regarding the cumulative tolerated dose or/and eliciting dose based on oral food challenge (OFC) results or previous allergic reactions.In our institution, the starting dose was usually set at 1/10 and lower of the eliciting dose.Whereas conventional oral immunotherapy is defined as continuing ingestion of allergenic food above the threshold, we set the initial and maintenance dose below the threshold for slow low-dose oral immunotherapy.In case children were able to continue taking the maintenance dose for several months, we did OFC to confirm the elevated eliciting or tolerated dose and increased the therapeutic dose slowly to the maintenance dose below the threshold.This procedure was repeated to increase the treatment dose as slow low-dose oral immunotherapy safely.We included cases where oral food challenge was not performed before the start of slow low-dose oral immunotherapy.The initial intake was determined in those children based on past allergic history.The treatment foods were orally administered.Children with slow lowdose oral immunotherapy took non-heated cow's milk or yogurt made with cow's milk (if the children needed to take a tiny amount of treatment food, they could take it as homemade pancakes or steamed bread) 12 at home.Treatment foods were determined by children's and caregivers' preferences based on personalized approaches and shared-decision making.Exclusion criteria were as follows: 1) loss to followup, 2) slow low-dose oral immunotherapy discontinuation of more than 3 months, and 3) with nonimmediate reactions.This study was approved by the institutional ethical review board (2022-057).The development modeling set included 120 children who first visited from January 2014 to December 2016.The validation modeling set included 71 children who first visited from January 2017 to October 2018.We defined the primary outcome as drinking 200 ml of cow's milk (6.6 g of cow's milk protein) in the first 3 years.Secondary outcomes were 10 ml cow's milk (0.33 g of cow's milk protein) intake in years 1, 2, and 3 after slow low-dose oral immunotherapy initiation.
We analyzed the comparison between 2 dataset groups using Student's t-test, Welch's t-test, or chisquare test.We reported the associations of specific parameters with primary outcomes using odds ratios (ORs) with 95% confidence intervals (CIs).A P value of <0.05 was defined to indicate statistical significance.All statistical analyses were performed with EZR and R version 4.1.3(The R Foundation for Statistical Computing, Vienna, Austria) for predictive modeling. 13 selected variables for developing predictive models from previously published reports and clinically important items.Models were as follows: Model 1: cow's milk-sIgE before slow low-dose oral immunotherapy þ age at beginning oral immunotherapy; Model 2: Model 1 þ TARC (thymus and activation-regulated chemokine), 14 serum biomarker of atopic dermatitis severity (commercially available in Japan), before slow low-dose oral immunotherapy initiation; and Model 3: Model 2 þ cow's milk -sIgE 1 year after slow low-dose oral immunotherapy.We created the models by logistic regression analysis using the development modeling dataset.A 3-fold crossvalidation was performed 100 times for internal validation and bootstrapped 2000 times to construct 95% confidence intervals for the receiver operating characteristic curve (ROC)-The Area Under the Curve (AUC).External validation was performed using calibration plots based on the validation modeling dataset.

Variable definitions
We used the capsulated hydrophilic carrier polymer (CAP) method to measure food antigenspecific antibody titers.This test method indicates a titer of 100 UA/ml or higher as "!100 UA/ml".All!100 UA/ml were analyzed for statistical analysis as 100 UA/ml.Severe atopic dermatitis was defined as EASI score >21.1 or SCORAD index >50.The grade of allergic symptoms was based on the severity classification of the anaphylaxis guidelines.

Handling of missing values
We did multiple imputations to perform completion on missing data and analyze with the maximum number of cases.Four Variables were used in multiple imputations: gender, presence of atopic dermatitis at first visit, total IgE level before the start of slow low-dose oral immunotherapy, and cow's milk-sIgE level before the start of slow low-dose oral immunotherapy.A biostatistician supported statistical analysis.

RESULTS
Of the 3480 children who visited the allergy center for the first time during the relevant period, 1011 were sensitized to cow's milk-sIgE (see Fig. 2).Of these, 262 children had eliminated dairy products at their first visit and had started slow low-dose oral immunotherapy for cow's milk under 4 years of age.A total of 191 (¼120 þ 71) children obtained cow's milk remission within 3 years or could be followed for 3 years.The clinical characteristics of the children are shown in Table 1.176 children (92.1%) had atopic dermatitis at the first visit.Table 2 demonstrates slow low-dose oral immunotherapy information.The median age in months at slow low-dose oral immunotherapy start was 19 months.The mean slow low-dose oral immunotherapy starting amount of cow's milk was 1.6 ml.On average, the children drank 28.8 AE 54.6 ml after 1 year, 69.3 AE 85.2 ml after 2 years, and 93.4 AE 93.0 ml after 3 years.After 3 years of slow low-dose oral immunotherapy, 61% of children could take 10 ml of cow's milk (0.33 g of cow's milk protein), and 40% could drink 200 ml of cow's milk (6.6 g of cow's milk protein).During slow low-dose oral immunotherapy, anaphylaxis was only observed 1 (0.83%) in the development dataset and 2 (2.8) % in the validation dataset.However, anaphylaxis related to accidental cow's milk intake, except slow low-dose oral immunotherapy, was more frequent than anaphylaxis events related to slow low-dose oral immunotherapy (see Table S1).Table S2 demonstrates the results of blood tests.Cow's milk-sIgE, before slow low-dose oral immunotherapy started, was 30.1 AE 33.8 UA/mL (mean AE SD).Tables 3 and 4 and Table S3 show the internal validation results with the adjusted model obtained from the development modeling set in Supplemental Figs. 3 and 4 For the secondary outcome (10 ml cow's milk [0.33 g of cow's milk protein] after 1, 2, and 3 years), Model 1 demonstrated AUC 0.80 (95%CI: 0.73-0.88)for 10 ml after 1 year, AUC 0.83 (95%CI: 0.76-0.90)for 10 ml cow's milk after 2 years, and AUC 0.81 (95%CI: 0.73-0.89)for 10 ml cow's milk after 3 years.Model 3 showed excellent discrimination with the AUC 0.83 (95% CI:0.76-0.91) on internal validation (see Table S3).Application of the model in the validation set gave an excellent discrimination (AUC 0.89, 95% CI:0.80-0.97)and a reasonable calibration (intraclass correlation coefficient [ICC] ¼ 0.88, 95% CI:0.62-0.97)(see Fig. 3).For secondary outcomes, internal validation and external application in the validation modeling set showed the results in Table S4 and Fig. 4.

DISCUSSION
We developed and validated predictive modeling for determining the remission rate 3 years CM-sIgE 1 year after OIT 0.916 (0.845, 0.993) 0.904 (0.842, 0.971) 0.928 (0.888, 0.97) Table 4. Multivariable associations between the predictors and the secondary outcome (drink 10 ml after 1, 2 or 3 year) in the development set (Model 3).CM: cow's milk, OIT: oral immunotherapy, OR: Odds ratio, CI: confidence interval  chemokine produced by epidermal keratinocytes, and serum TARC levels reflect the severity of atopic dermatitis.TARC is correlated with SCORAD score, so a higher serum TARC level indicates that atopic dermatitis is more severe.6][17] As we mentioned before, improved eczema management itself could reduce IgE titers.In our previous randomized controlled trial (RCT) investigating immune tolerance induction for infants, poorly controlled atopic dermatitis infants they developed hen's egg allergy even though they took a tiny dose of hen's egg.A recent RCT also demonstrated that eczema management via skin intervention could reduce hen's allergy in infants. 18Exposures to peanuts in dust and sensitization levels differed among eczema and non-eczema children. 19Based on the dual allergen hypothesis, eczema management via skin and oral allergenic intervention is also necessary for tolerance induction in treating food allergy and preventing food allergy. 20Various food allergens such as peanuts, hen's egg, and walnuts are in our environment, 19,21,22 so the skin barrier is also crucial to prevent percutaneous IgE sensitization via inflamed skin.Before oral immunotherapy, children need to keep eczema controlled well.Our model could show eczema management's importance in children with atopic dermatitis and food allergy.
As for including the variable of IgE sensitization, our study results coincided with a past study 23 since Peterson et al demonstrated that the level of sIgE had a greater risk predictive factor for cow's milk remission. 23e empirically understand that cow's milk children with a significant decline in cow's milk-sIgE about 1 year after initiation of oral immunotherapy can achieve early remission from clinical experience.We included cow's milk-sIgE one year after initiation of slow low-dose oral immunotherapy as a variable in our model and provided accuracy and fit in the predictive model.We consider that IgE titer trajectory at the one-year point after the start of treatment is valuable to capture the future prognosis of cow's milk allergy by oral immunotherapy.
In consultation with biostatisticians, we selected variables from previously published reports and clinically important items.Because there are multiple factors associated with food allergy prognosis, in this study, we aim to combine several variables to create a model and then add several more variables to make the prediction more accurate.Since we do not have a large sample size, we would like to examine which factors are more influential in the subsequent study using extensive data from future studies.We believe this prediction modeling is helpful before we discuss with children and families their future cow's milk allergy prognosis through slow low-dose oral The strength was that slow low-dose oral immunotherapy was safe, and anaphylaxis was rare.Children and families could safely be satisfied with slow low-dose oral immunotherapy introduction without anxiety.However, we have several limitations of this study.First, this was a retrospective study and a single-center study.Now, we have an ongoing prospective observational cohort study for food allergy.Second, the detailed slow low-dose oral immunotherapy regimen was decided based on shared-decision making, but our data reflected a real-world clinical practice.Third, we focus on only cow's milk allergy in this study but would like to develop another predictive model for hen's egg, peanut, and tree nuts allergies.Fourth, although TARC 24 is approved as a clinical test in Japan, it is not globally available in clinical practice.In this study, because of the large number of missing EASI or SCORAD variables, we chose TARC.Since TARC was highly correlated with SCORAD, 24 we assumed that the accuracy of the predictive model would be equivalent even if the variables were replaced from TARC to SCORAD.We hope to release the next version with SCORAD as variables for practical use.

CONCLUSION
We developed and validated predictive models for determining the remission rate three years after slow low-dose oral immunotherapy initiation at less than four years of age in young children with cow's milk allergy.This model is highly accurate and may help manage cow's milk allergy.

Fig. 2
Fig.2Flow chart of the study

Fig. 3
Fig.3Calibration plots in Models 1, 2 and 3 for the primary outcome.Model-specific calibration plots in the external validation of the predictive model with the primary outcome (200 ml cow's milk intake three years later)

Fig. 4
Fig. 4 Calibration plots in Models 1, 2 and 3 for the secondary outcomes.Calibration plots for external validation in Model 3 to predict secondary outcomes (drinking 10 ml cow's milk within 1, 2 and 3 years)

Table 1 .
Characteristics of the children in this study.
AD: atopic dermatitis, FA: food allergy, CM: cow's milk, OIT: oral immunotherapy, OFC: oral food challenge, OAS: oral allergy syndrome, SD: standard deviation.aInformation at the time of the initial visit.bThose that meet EASI>21.1 or SCORAD>50after slow low-dose oral immunotherapy under 4 years of age in children with cow's milk allergy.This predictive model is highly accurate and can support cow's milk allergy management.Variables included in the model (Cow's milk-sIgE before slow low-dose oral immunotherapy þ age at beginning slow low-dose oral immunotherapy þ TARC (eczema severity marker) before slow low-dose oral immunotherapy initiation þ cow's milk-sIgE 1 year after slow low-dose oral immunotherapy) were reasonable.Future prognosis is influenced not only by a single factor but multiple factors such as age, sIgE
OIT: oral immunotherapy, SD: standard deviation, AR: allergic reaction, AI: accidental ingestion.aIf not one, the main one used titers, eczema management, Etc.We need to consider various factors that affect the efficacy and future prognosis of oral immunotherapy.One of the novel points in this study is that we included atopic dermatitis severity (TARC) in our modeling with excellent accuracy.TARC is a type of

Table 3 .
Multivariable associations between the predictors and the primary outcome in the development set (Model 3).CM: cow's milk, OIT: